This invention relates in general to the field of flow cytometer systems for studying characteristics of a stream, including particles entrained in droplets of a stream, and particularly for monitoring and controlling droplet sorting and specifically droplet generation. The sorting of biological cells entrained in streams and the droplets thereof provides relatively pure samples for both biomedical research and clinical medicine. Various techniques of sorting cells using flow cytometry have been employed over the last two decades. See, e.g., Lindmo et al., “Flow Sorters for Biological Cells” in Flow Cytometry and Sorting, 2nd ed. Wiley-Liss, Inc.: New York, 1990, pages 145-169; and U.S. Pat. Nos. 3,710,933; 4,361,400; 4,981,580; 5,483,469; and 5,602,039, among others.
Techniques used for cell sorting include flow sorters, such as flow cytometers. Conventional flow cytometers, such as illustrated in FIG. 1 of U.S. Pat. No. 6,079,836, include apparatus for generating a flow of a fluid consisting of small amounts of sample (which may be dye-treated) in a sheath fluid. Generally, the sheath fluid and its entrained sample are hydrodynamically focused and exit a nozzle, thereby forming a jet stream. A droplet generator in the cytometer generates droplets from the stream, such as by causing the jet to oscillate, thereby forming individual droplets for sorting. Additionally, conventional cytometers contain an apparatus for charging the jet to enable deflection of the droplets. See, e.g., U.S. Pat. Nos. 5,602,039 and 5,483,496. The cytometers also contain a device for deflecting the droplets and creating post-deflection trajectories of the droplets that are a function of the charge. Additionally, such conventional cytometers contain a collection apparatus for collecting the droplets having common post-deflection trajectories.
A crucial component of such cytometers and the systems by which they operate are the components that enable detection and imaging of characteristics of the stream, and particularly the droplets. For example, light beams or lasers can be directed at the sheath and sample stream, which can include the droplets, to excite and fluoresce dyes in the stream. Various parameters, such as forward and side light scatter, and fluorescent wavelengths of light are detected to identify sample particles in the stream and to sort specific droplets to obtain samples of the particles therein. Still other cytometers contain standard video cameras and standard video capture systems, such as charge coupled device (CCD) cameras, and other video apparatus to capture images of the stream and droplets.
Such conventional flow cytometers consist of either an electrostatic sorting system or a fluidic sorting system that is used to sort the particles. The electrostatic system enables control of certain sorting parameters by the system operator. The electrostatic sorting parameters that the operator controls include droplet charge, crystal frequency, crystal drive amplitude, sheath pressure and sample pressure.
Typically, sorting of the droplets containing the sample particles is accomplished over an extended period of time during which environmental changes can adversely influence the performance of an electrostatic sorter. Such environmental changes include temperature, acoustic vibrations, and atmospheric pressure. One or more of these environmental changes continually affects the sort, requiring the operator of the system to adjust the instrument during sorting to avoid contamination of the sort. To keep sort efficiency high, operators trained in the adjustment of the instrument must monitor the appearance of the jetting stream, and manually correct for any changes. It is both costly and tedious for an operator to be constantly monitoring these parameters and the sort performance.
Solutions of the prior art for improving the sort have included, among others, the use of a direct jet monitor, such as an optical monitor to illuminate the jet and the conditions within the jet through a charge coupled device (CCD) camera. Cytometers using this system provide an optical image of the actual droplet separation point and jet stream. Some optical monitoring systems are associated with a feedback system to automatically change the horizontal location of the jet or the point at which droplet separation occurs. See, e.g., U.S. Pat. No. 5,602,039. Still other cytometer systems examine the drop delay time based on a measurement of the speed of the fluid in the stream or determine the width of the stream correlative to the nozzle size used by the cytometer. See, e.g., U.S. Pat. No. 6,248,590.
Still other improved flow cytometers of the art excite the fluid jet by an acoustic vibration of 5 to 200 KHz, typically produced by a piezo crystal element, although any vibrating device can be used. This excitation can cause the jet stream to undulate and produce droplets at the rate of excitation. The phase of the undulations and the droplets produced, as well as the position of the undulations and droplets in reference to the jet stream nozzle are critical to the proper operation of these sorters. In order to automate control of the undulations and phase, images of the jetting stream are captured using a standard video camera and a standard video capture system.
However, an automation system utilizing video images and video processing requires that the jetting stream have sufficient contrast to be analyzed. Current imaging means produce an image with little contrast between the jetting stream and the light field behind the jetting stream. Other solutions in the art for improving sorting include the use of hardware such as image intensifiers and analog video processors to improve the contrast prior to digitization. However, the amount of contrast improvement is limited, and additional image manipulation can remove detail and alter the true image so that proper control is not ideal. See, e.g., U.S. Pat. No. 5,700,692.
Therefore, a need in the art exists for efficient methods, systems, and apparatus for maintaining a stable sorting system by establishing a stable sorting system and automatically adjusting sorting parameters of the sorting system. A need further exists for compositions and methods that allow the effects of the adjustments to be monitored and further allow that adjustment of these parameters to maintain a stable sorting system. Enhancements to flow cytometer apparatus and methods to enable contrast improvement and allow accurate characterization of particles in the droplet are also needed.